PROJECT SUMMARY Membrane protein recycling through the endolysosomal network plays a critical role in maintaining cellular homeostasis. Perturbations in the sorting mechanisms have been linked to human disease including cancer. In this regard, we discovered the CCC complex (COMMD/CCDC22/CCDC93) which regulates both Retromer and Retriever dependent cargo trafficking. Retromer and Retriever are two structurally and functionally related complexes that are involved in cargo selection and are responsible for the trafficking of cargos either to the lysosomes for degradation or to the cell surface or trans-Golgi network for recycling. Retriever is composed of VPS35L, VPS26C and VPS29. VPS35L is a shared subunit between the CCC and retriever complexes. The role that this system may play in carcinogenesis is only incompletely understood at this point. Previous studies from our laboratory found that COMMD1 expression is frequently repressed in various tumors and this repression promotes tumor invasion. Furthermore, by analyzing the data accessible through cBioPortal, I found that VPS35L is significantly downregulated or mutated in various tumors, particularly hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC), which promoted us to examine the functional consequence of tumor-associated missense mutations in this gene. Data obtained through my K01 proposal suggest that the loss of VPS35L leads to endosomal trapping of multiple cargos such as Integrins and Notch2, further resulting in their decreased expression on the cell surface. Further, HCC-associated mutations in VPS35L gene behave as loss-of-function variants. However, my preliminary data suggest that in regards to EGFR trafficking, VPS35L loss results in increased accumulation of EGFR at the plasma membrane. Detailed mechanism underlying the regulation of EGFR trafficking by VPS35L is unknown. In this regard, a recent study suggested that FAM45A, a protein that associates with the CCC complex and Retriever, regulates EGFR transition from early to late endosomes. Thus, the central hypothesis of my studies is that CCC and Retriever regulate the transition of specific endosomal cargos, such as EGFR, to destinations other than the plasma membrane such as the late endosome. The project?s overall goal is to demonstrate the mechanism by which Retriever regulates EGFR trafficking under physiologic conditions and to determine the functional impact of colon cancer associated mutations and to address this goal I propose the following specific aims: (1) Determine the specific steps in EGFR trafficking that are regulated by VPS35L. (2) Examine the functionality of colon cancer-associated VPS35L mutations. The proposed studies combine biochemical and immunofluorescence approaches, and state-of-the-art technologies for molecular analysis. Altogether, this work will establish a fundamental understanding in the regulation of endosomal sorting, which will have important basic cell biology, as well test the direct relevance of this pathway in colon carcinogenesis.